PROJECT SUMMARY Pathology stemming from excess ectopic bone formation, or trauma-induced heterotopic ossification (HO), presents a substantial barrier to recovery in 20% of patients with hip replacements, musculoskeletal trauma, spinal cord injury, amputations and burn injuries. Patients with HO experience chronic pain, restricted joint function, and open wounds; they often undergo surgical procedures to excise the offending bone, but these procedures fail to reverse the joint contractures and restricted range of motion. Even after a successful excision procedure, recurrence is common. With these current limitations in our understanding of HO and our inability to prevent its development, we set out to clarify the cells responsible and primary signaling pathways involved. Furthermore, we plan to validate a novel cell specific drug delivery system to block secretion of a primary ligand (Bone Morphogenetic Protein-2) from a primary cell (macrophage) responsible for inducing HO. Analysis of patients at high risk for HO and of our burn/tenotomy HO model have demonstrated an increase in Hypoxia Inducible Factor 1alpha (HIF-1?) and Bone Morphogenetic Protein 2 (BMP2) signaling. However, where the BMP2 ligand originates and how to block its effect without causing off-target effects is unknown. Additionally, our data show that macrophages play a central role in HO and further studies are needed to elucidate how they are recruited and what ligands they secrete when they traffic to the injury site. The following aims are to test our hypothesis that that inhibition of mesenchymal cell HIF-1? mediated macrophage recruitment or inhibition of macrophage Bmp2 expression will prevent trauma-induced HO. Aim 1: To demonstrate that genetic loss of Hif1a reduces SDF1 production by mesenchymal cells and macrophage infiltration after injury. This aim will demonstrate that Hif-1?-mediated production of SDF-1 by mesenchymal cells at the injury site is responsible for macrophage recruitment immediately following injury. Aim 2: To define the role of macrophage recruitment and macrophage-specific production of BMP2 on HO. This aim will demonstrate that macrophage migration to the injury site and macrophage production of BMP2 is critical for ectopic mesenchymal cell chondrogenesis and heterotopic ossification. Aim 3: To demonstrate that novel microparticles can be used to silence genes specifically in macrophages. This aim will optimize microparticles for macrophage-specific uptake and drug delivery to administer Bmp2 siRNA.